We have investigated the role of membrane CPE and secretogranin III as sorting receptors for targeting POMC to the regulated secretory pathway(RSP). Using our CPE knockout (KO) mouse, we showed that 50% of newly synthesized POMC in primary cultures of the pituitary anterior lobe cells was degraded and suggests that in the absence of efficient sorting to the granules of the RSP due to the lack of CPE, POMC was targeted for degradation. However, some of the remaining POMC was sorted into the RSP. A candidate for a compensatory sorting receptor is Secretogranin III (SgIII), which has been shown to bind POMC in precipitation assays. SgIII, is a member of the granins that are found in neuroendocrine cells and is involved in trafficking of chromogranin A (CgA) to the RSP. We used RNA interference (siRNA) to knock down SgIII and CPE expression in AtT20 cells and demonstrated that increased POMC was secreted via the constitutive secretory pathway in both cases. Increased constitutive secretion of CgA was only observed in the SgIII knockdown cells. In double CPE-SgIII knock down cells, increased constitutive secretion of POMC was observed and stimulated secretion of ACTH was perturbed. These results demonstrate that CPE is involved in the trafficking of POMC to the RSP;and that SgIII may play a compensatory role for CPE in the sorting of POMC to the RSP in addition to a more general role in the RSP trafficking process. Transport of hormone and BDNF vesicles to the plasma membrane for activity-dependent secretion is critical for endocrine function and synaptic plasticity. We showed that the cytoplasmic tail of a transmembrane form of CPE in hormone or BDNF-containing dense core secretory vesicles plays an important role in their transport to the release site. Overexpression of the CPE cytoplasmic tail in the cytoplasm to compete with the endogenous tail diminished localization of endogenous POMC, BDNF and fluorescence-tagged CPE in the processes of an endocrine cell line, AtT20;and hippocampal neurons. In hippocampal neurons, primary pituitary and AtT20 cells, overexpression of the CPE tail inhibited the movement of BDNF- and POMC/CPE-containing vesicles to the processes, respectively. S-tagged CPE tail pulled down microtubule-based motors, dynactin (p150), dynein and KIF1A/KIF3A from cytosol of AtT20 and brain cells. Finally, overexpression of the CPE tail inhibited the regulated secretion of ACTH from AtT20 cells. We also showed that the CPE tail interacted with C-terminus of gamma-adducin, a component of the cytoskeleton that binds and stabilizes F-actin. Overexpression of the C-terminal 38 amino acid of gamma-adducin inhibited the transport of POMC vesicles out of the cell body into the processes of AtT-20 cells. Thus these studies demonstrate that the vesicular CPE cytoplasmic tail plays a novel mechanistic role in anchoring regulated secretory pathway POMC/ACTH and BDNF vesicles to actin via gamma-adducin for movement immediately after budding from the TGN which is actin-based, and subsequently to the microtubule-based motor system for transport along the processes to the plasma membrane for activity-dependent secretion in endocrine cells and neurons. We recently found that transmembrane CPE is not only associated with large dense core vesicles (LDCVs), but also with glutamate-containing synaptic vesicles (SVs) in mouse hypothalamus and synaptic-like microvesicles in PC12 cells. High K+ stimulated release of glutamate from hypothalamic neurons was diminished in CPE-KO mice. Electron microscopy revealed that the number of SVs located in the pre-active zone (within 200nm of the plasma membrane at the active zone) of synapses was significantly decreased in hypothalamic neurons of CPE-KO mice compared with wild-type mice. Total internal reflective fluorescence (TIRF) microscopy using PC12 cells as a model showed that overexpression of the CPE cytoplasmic tail reduced the steady-state level of synaptophysin-containing synaptic-like microvesicles accumulated in the area within 200 nm from the sub-plasma membrane (TIRF zone). Our findings show that the CPE cytoplasmic tail, which interacts with gamma adduccin and actin, is a new mediator for the localization of SVs in the actin-rich pre-active zone in hypothalamic neurons and the TIRF zone of PC12 cells. Our recent studies in pituitary AtT-20 cells have provided evidence for an autocrine mechanism for up-regulating LDCV biogenesis to replenish LDCVs following stimulated exocytosis of these vesicles. The autocrine signal was identified as serpinin, a novel 26 amino acid CgA-derived peptide cleaved from the C-terminal of CgA. Serpinin was first isolated from AtT20 cell conditioned medium and demonstrated to be released in an activity-dependent manner from LDCVs. Subsequently, secreted serpinin was found to activate adenyl cyclase to increase cAMP levels, and protein kinase A in the cell. This then led to the translocation of the transcription factor sp1 from the cytoplasm into the nucleus and an increase in transcription of a protease inhibitor, protease nexin 1 (PN-1), which then inhibited granule protein degradation in the Golgi complex. The stabilization of those proteins increased their levels in the Golgi, resulting in significantly enhanced LDCV formation. CPE plays a significant role in obesity, and recently the gene has been coined an obesity susceptibility gene. We showed that CPE KO mice were not able to process pro-CART to CART and therefore lacked this anorexigenic neuropeptide, in the hypothalamus. These animals over-eat and become obese, thus providing further evidence linking decrease of this neuropeptide to the cause of obesity. Additionally, in collaboration with the Accili group at Columbia University, it was found that the transcription factor FoxO1 negatively regulates CPE gene expression. Normally insulin binds to insulin receptors in the POMC neurons and that leads to nuclear signaling, nuclear exclusion and inactivation of FoxO1. To model this physiological event, FoxO1 was deleted in the POMC neurons in the arcuate nucleus of the hypothalamus in mice and that resulted in increased CPE levels, increased alpha-MSH, an anorexigenic neuropeptide derived from POMC, and reduced food intake without change in energy expenditure. These findings raise the possibility of targeting CPE to develop weight loss medications. We also showed that extremely obese CPE-KO mice have low bone mineral density and concluded that the lack of CART which promotes bone formation, is an important player responsible for poor bone density in these mice. CPE-KO mice have deficiencies in the nervous system. Morris water maze and object preference tests indicate a problem with learning and memory. We showed that in 6-14 week old CPE-KO mice, dendritic pruning was poor in cortical and hippocampal neurons which would affect synaptogeneis. Additionally electrophysiological measurements showed a defect in the generation of long term potentiation (LTP) in hippocampal slices of these mice. A major cause for this defect was due to the loss of neurons in the CA3 region of the hippocampus of CPE KO animals observed at 4 weeks of age and older. These neurons, which are normally enriched in CPE, were normal at 3 weeks of age just before the animals were weaned. Interestingly, when weaning was delayed a week, this degeneration was not observed till postnatal week 5 in the CPE KO mice. These results suggest that the degeneration is correlated with the stress of weaning and maternal separation and that CPE is important in maintaining the survival of CA3 neurons during that period. Indeed, we showed that when CPE was overexpressed in hippocampal neurons in culture, they were protected from apoptosis after induced oxidative stress using hydrogen peroxide. Thus, CPE has a novel neuroprotective role in hippocampal neurons.